Multiplexed assays are used in several fields of technology to simultaneously detect multiple targets (e.g., dozens or more) in a single procedure where the presence and/or the activity of the targets is determined quantitatively or qualitatively.
Multiplexed assay detection is typically performed by measuring a signal, such as fluorescence and chemoluminescence, and determining the outcome of the assay based on the position of the detected signal with respect to a predetermined threshold.
In particular, the interpretation of the results of a multiplexed assay typically begins by comparing a raw response to a threshold value. Signal detection theory has been used to describe the effect of varying threshold value on assay performance [see references 15, 16]. An ideal threshold value completely separates positive sample responses from negative sample responses In practice, however, distributions of responses generated by positive and negative samples can overlap. When overlap occurs, the number of both true positives (positive samples exceeding threshold) and false positives (negative samples exceeding threshold) increases as the threshold value decreases.
Therefore, accurate interpretation of multiplexed assays is currently a challenging task, in particular with reference to the correct determination of quantitative detection of multiple targets.